Project Summary/Abstract Now that protein folding is becoming better understood, we can study it in combination with other interactions that proteins make with biomolecules. Proteins in cells and organisms continuously interact with other biomolecules, such as RNA. They are also modified with attachments, such as polyethylene glycol (PEG) molecules that can enhance stability for drug delivery. Finally, domains of larger proteins can interact with one another, modifying the folding process or leading to undesirable aggregation, which can lead to protein diseases. Our long-term objective is to study interactions of proteins with PEG, RNA, and other protein domains, and to characterize these interactions quantitatively. Within that long-term objective, our specific aims are threefold: 1) PEG is used extensively in the pharmaceutical industry to improve the delivery of protein drugs. We study how PEG interacts with protein surfaces, so we can figure out the mechanism by which PEG helps stabilize protein drugs for delivery. We will study several protein systems, including a therapeutic agent for chronic kidney disease. 2) The spliceosome assembles in the cell nucleus to splice and re-assemble messenger RNA, which is necessary to take the information to make new proteins from the nucleus to the ribosomes, where proteins are synthesized. We will study one of the key protein-RNA interactions by making many mutants and comparing them with a new model we just developed, that we think can predict how strongly protein and RNA will bind. This will be important for rational design of drugs to interfere with, or repair, protein-RNA interactions. 3) Large proteins contain many domains, and when they fold things can go wrong. We study these interactions in an expanded phase diagram of pressure and temperature, to better understand their physical origins. We discovered that folding intermediates, which are structures that are not quite properly folded, can appear and disappear in this phase diagram. By learning why this happens we can better suppress such intermediates, which could form harmful aggregates. To achieve our goals, we are developing new fluorescence assays to rapidly and sensitively detect interactions. We are expanding the capabilities of our protein pressurization techniques, so we can study protein under conditions relevant to pressure sterilization of food. And we are making new PEG-labeled proteins to study how important PEG length and attachment sites really are.